Sitagliptin plus pantoprazole can restore but not maintain insulin independence after clinical islet transplantation: results of a pilot study.

AIMS: Resuming insulin use due to waning function is common after islet transplantation. Animal studies suggest that gastrointestinal hormones, including gastrin and incretins may increase ß-cell mass. We tested the hypothesis that pantoprazole plus sitagliptin, would restore insulin independence in islet transplant recipients with early graft insufficiency and determined whether this would persist after a 3-month washout. METHODS: Single-centre, uncontrolled, open label study of sitagliptin 100 mg daily plus pantoprazole 40 mg twice daily for 6 months. RESULTS: After 6 months of treatment, two of eight participants (25%) achieved the primary endpoint, defined as HbA1C < 42 mmol/mol (6%), fasting plasma glucose < 7.0 mmol, C-peptide > 0.5 nmol and no insulin use. There was a significant reduction in mean insulin dose, but no change in HbA1C or weight. There were no changes in the acute insulin response to arginine, the mixed meal tolerance test or blinded continuous glucose monitoring. After the washout, no participants met the primary endpoint and HbA1C increased from 45 ± 8 mmol/mol (6.3 ± 0.7%) to 51 ± 6 mmol/mol (6.8 ± 0.6%) (P < 0.05). Two participants had mild-moderate transient gastrointestinal side effects. There were no episodes of hypoglycaemia. CONCLUSIONS: Sitagliptin plus pantoprazole is well tolerated and safe and may restore insulin independence in some islet transplant recipients with early graft insufficiency, but this was not sustained when treatment was withdrawn. A larger, controlled trial is required to confirm the effectiveness of this combination to achieve insulin independence and to confidently exclude any persistent benefit for graft function. (Clinical Trials Registry No.: NCT00768651).

Validation of the BETA-2 Score: An Improved Tool to Estimate Beta Cell Function After Clinical Islet Transplantation Using a Single Fasting Blood Sample.

The beta score, a composite measure of beta cell function after islet transplantation, has limited sensitivity because of its categorical nature and requires a mixed-meal tolerance test (MMTT). We developed a novel score based on a single fasting blood sample. The BETA-2 score used stepwise forward linear regression incorporating glucose (in millimoles per liter), C-peptide (in nanomoles per liter), hemoglobin A1c (as a percentage) and insulin dose (U/kg per day) as continuous variables from the original beta score data set (n = 183 MMTTs). Primary and secondary analyses assessed the score's ability to detect glucose intolerance (90-min MMTT glucose ≥8 mmol/L) and insulin independence, respectively. A validation cohort of islet transplant recipients (n = 114 MMTTs) examined 12 mo after transplantation was used to compare the score's ability to detect these outcomes. The BETA-2 score was expressed as follows (range 0-42): [Formula: see text] A score <20 and ≥15 detected glucose intolerance and insulin independence, respectively, with >82% sensitivity and specificity. The BETA-2 score demonstrated greater discrimination than the beta score for these outcomes (p < 0.05). Using a fasting blood sample, the BETA-2 score estimates graft function as a continuous variable and shows greater discrimination of glucose intolerance and insulin independence after transplantation versus the beta score, allowing frequent assessments of graft function. Studies examining its utility to track long-term graft function are required.

Differential gene regulation by 9-cis and all-trans retinoic acid in neuroblastoma cells.

BACKGROUND: 9-cis retinoic acid (RA) is more effective than all-trans RA at inducing neuroblastoma differentiation in vitro, and has distinct biological properties with respect to its ability to promote apoptosis in N-type neuroblastoma cells. The cellular effects of 9-cis RA may, in part, result from activation of retinoid X receptor (RXR) homodimers. If this hypothesis is correct, 9-cis RA may control the expression of a different subset of retinoid-regulated genes compared to all-trans RA. PROCEDURE: We have therefore used differential mRNA display to identify genes differentially expressed in neuroblastoma cells in response to all-trans and 9-cis RA. RESULTS: The majority of cDNAs differentially expressed in response to all-trans or 9-cis RA matched to nonredundant Genbank sequences or EST database sequences. Differential-display profiles were similar in SH SY 5Y and SH S EP cells, clonal derivatives of the mixed neuroblastoma cell line SK N SH, although there were apparent differences between these cell lines with respect to the retinoid-regulation of specific RT-PCR cDNA fragments. CONCLUSIONS: These data support the view that 9-cis and all-trans RA act via different receptor mechanisms.

p53 cellular localization and function in neuroblastoma: evidence for defective G(1) arrest despite WAF1 induction in MYCN-amplified cells.

This study investigated the hypothesis that p53 accumulation in neuroblastoma, in the absence of mutation, is associated with functional inactivation, which interferes with downstream mediators of p53 function. To test this hypothesis, p53 expression, location, and functional integrity was examined in neuroblastoma by irradiating 6 neuroblastoma cell lines and studying the effects on p53 transcriptional function, cell cycle arrest, and induction of apoptosis, together with the transcriptional function of p53 after irradiation in three ex vivo primary, untreated neuroblastoma tumors. p53 sequencing showed five neuroblastoma cell lines, two of which were MYCN-amplified, and that all of the tumors were wild-type for p53. p53 was found to be predominantly nuclear before and after irradiation and to up-regulate the p53 responsive genes WAF1 and MDM2 in wild-type p53 cell lines and a poorly-differentiated neuroblastoma, but not a differentiating neuroblastoma or the ganglioneuroblastoma part of a nodular ganglioneuroblastoma in short term culture. This suggests intact p53 transcriptional activity in proliferating neuroblastoma. Irradiation of wild-type p53 neuroblastoma cell lines led to G(1) cell cycle arrest in cell lines without MYCN amplification, but not in those with MYCN amplification, despite induction of WAF1. This suggests MYCN amplification may alter downstream mediators of p53 function in neuroblastoma.

Evidence for the development of p53 mutations after cytotoxic therapy in a neuroblastoma cell line.

p53 mutations are rare in neuroblastomas at diagnosis perhaps accounting for their initial response to therapy, but advanced neuroblastoma frequently relapses, and it is possible that p53 mutations develop later. Two neuroblastoma cell lines derived from the same patient before [SKNBE(1n)] and after [SKNBE(2c)] cytotoxic therapy were analyzed for the presence of chromosome 17 and p53 genes by fluorescent in situ hybridization, p53 mutations by DNA sequencing, and p53 function after irradiation by studying the transcription of p53-regulated genes, cell cycle arrest, and induction of apoptosis. The SKNBE(1n) cell line was wild-type for p53, had two p53 genes, two copies of chromosome arm 17p and showed functional p53 after irradiation. The SKNBE(2c) cell line derived from the same patient 5 months later at relapse had loss of an entire chromosome 17, resulting in hemizygosity for the p53 locus on 17p and a missense p53 mutation in exon 5, and p53 was not functional after irradiation. The appearance of a p53 mutation in a cell line derived from a relapsed neuroblastoma suggests that this may be a mechanism of resistance to therapy. If p53 mutations develop frequently in relapsed neuroblastoma, cytotoxic agents more sensitive to mutant p53 might be more effective at relapse.

Distinct properties of fenretinide and CD437 lead to synergistic responses with chemotherapeutic reagents.

The RARbeta/gamma-selective retinoids fenretinide and CD437 induce caspase-dependent apoptosis but generate free radicals independently of caspases. Apoptosis, but not free radical generation, induced by these retinoids is inhibited by RARbeta/gamma-specific antagonists. Both fenretinide and CD437 induce apoptosis synergistically with cisplatin, carboplatin, or etoposide. However, antioxidants inhibit this synergy to the level obtained with chemotherapeutic drugs alone, and this implies that free radical generation is important in the synergistic response. Since apoptosis induced by fenretinide or CD437 is mediated by apoptotic pathways involving RARs and/or mitochondria and differs from mechanisms of chemotherapy-induced apoptosis this may explain the strong synergistic response seen between these synthetic retinoids and chemotherapeutic drugs. These results suggest that fenretinide or CD437 may be useful adjuncts to neuroblastoma therapy.

Synergistic induction of apoptosis of neuroblastoma by fenretinide or CD437 in combination with chemotherapeutic drugs.

Retinoic acid therapy improves the survival of children with neuroblastoma and 13-cis retinoic acid now forms an important component of treatment for residual disease of stage IV neuroblastoma after chemotherapy. However, although 13-cis retinoic acid induces differentiation, other retinoids are effective at inducing apoptosis of neuroblastoma in vitro, including the novel compounds fenretinide and CD437 and these may be alternative retinoids for neuroblastoma therapy. The aim of our study was to evaluate the ability of fenretinide, CD437 (6-¿3-(1-adamantyl)-4-hydroxyphenyl¿ -2-naphthalene carboxylic acid) and different retinoic acid isomers to induce apoptosis of neuroblastoma in conjunction with the chemotherapeutic drugs, cisplatin, etoposide and carboplatin. Neuroblastoma cell lines were treated with retinoids prior to treatment with chemotherapeutic agents and flow cytometry used to measure apoptosis and free radical generation. Pre-treatment of neuroblastoma cell lines with fenretinide or CD437 prior to treatment with cisplatin, etoposide or carboplatin synergistically increased apoptosis, an effect not seen with 13-cis, all-trans or 9-cis retinoic acid. Contrary to retinoic acid isomers or chemotherapeutic drugs, apoptosis of neuroblastoma cells induced by fenretinide or CD437 was accompanied by the generation of intracellular free radicals. Quenching of fenretinide- or CD437-induced free radicals with antioxidants abolished the synergistic response seen with the subsequent addition of chemotherapeutic agents. Therefore, the generation of free radicals by fenretinide or CD437 may be the key property of these retinoids leading to synergistic responses with chemotherapeutic drugs. Clearly, these synthetic retinoids provide new opportunities for novel neuroblastoma therapy.

Giant ancient schwannoma of the pelvis.

A 43-year-old man with a large ancient schwannoma of the pelvis, presenting with varicose veins, is reported. Ancient schwannoma (neurilemmoma) is a benign tumour of nerve sheath origin characterised histologically by features of severe degeneration and which rarely can grow to a large size. Malignant transformation, though reported, is extremely rare.

Effector mechanisms of fenretinide-induced apoptosis in neuroblastoma.

Fenretinide is an effective inducer of apoptosis in many malignancies but its precise mechanism(s) of action in the induction of apoptosis in neuroblastoma is unclear. To characterize fenretinide-induced apoptosis, neuroblastoma cell lines were treated with fenretinide and flow cytometry was used to measure apoptosis, free radical generation, and mitochondrial permeability changes. Fenretinide induced high levels of caspase-dependent apoptosis accompanied by an increase in free radicals and the release of cytochrome c in the absence of mitochondrial permeability transition. Apoptosis was blocked by two retinoic acid receptor (RAR)-beta/gamma-specific antagonists, but not by an RARalpha-specific antagonist. Free radical induction in response to fenretinide was not blocked by the caspase inhibitor ZVAD or by RAR antagonists and was only marginally reduced in cells selected for resistance to fenretinide. Therefore, free radical generation may be only one of a number of intracellular mechanisms of apoptotic signaling in response to fenretinide. These results suggest that the effector pathway of fenretinide-induced apoptosis of neuroblastoma is caspase dependent, involving mitochondrial release of cytochrome c independently of permeability changes, and mediated by specific RARs. As the mechanism of action of fenretinide may be different from other retinoids, this compound may be a valuable adjunct to neuroblastoma therapy with retinoic acid and conventional chemotherapeutic drugs.

Incidence and survival for cancer in children and young adults in the North of England, 1968-1995: a report from the Northern Region Young Persons' Malignant Disease Registry.

The Northern Region Young Persons' Malignant Disease Registry records information on young people under 25 years old diagnosed with cancer in the Northern Region of England. Incidence and survival rates were calculated for children and young adults diagnosed with cancer between 1968 and 1995. There were 2099 (M:F 1.28:1) children (age 0-14 years) and 2217 (M:F 1.23:1) young adults (15-24 years) diagnosed with a first cancer between 1968 and 1995. The age-standardized rate (ASR) for childhood cancer was 121 per million 0 to 14 year-olds per year. For young adults the ASR was 175 per million 15 to 24 year-olds, per year. Incidence of childhood cancer increased over time at a rate of 12 extra cases per million children, per decade (P < 0.001). In young adults incidence rates increased by 16 extra cases per million 15 to 24 year-olds, per decade (P < 0.001). For childhood cancer 5-year survival was 42% for those diagnosed 1968-1977, 57% for 1978-1987 and 71% (95% CI 67-75) for 1988-1995. Survival for young adults over the three periods was 45%, 62% and 73% (95% CI 70-78) respectively. The cumulative risk of developing cancer before the age of 25 is 1 in 285. Over the 28-year period there were significant improvements in survival and modest increases in incidence in both children and young adults.

Fluorescence in situ hybridization techniques for the rapid detection of genetic prognostic factors in neuroblastoma. United Kingdom Children's Cancer Study Group.

Neuroblastoma is the commonest extracranial solid tumour in children. There are a number of molecular genetic features known which are of prognostic importance and which are used to direct therapy. Identification and targeting of high-risk individuals with intensive therapeutic regimens may allow an improvement in survival rates. The most powerful biological parameters associated with prognosis in this malignancy are chromosomal changes, especially MYCN amplification, deletion of chromosome 1p and aneuploidy. Rapid characterization of these aberrations at the time of diagnosis is paramount if stratification according to risk group is to be achieved. This paper describes the rapid detection of del(1p), MYCN amplification and trisomy using interphase fluorescence in situ hybridization on imprints from fresh tumour biopsies. The results are related to those obtained by standard molecular methods and karyotyping.

A technique for the evaluation of failed fallopian tube ligation with metal clips.

The evaluation of fallopian tubes after failed tubal ligation can be difficult because conventional histopathological techniques are unable to section the metal clips when in situ. Once the clips have been removed, any evidence of tube patency is lost. This report describes a technique of embedding and sectioning that enables sections to be made while the metal clips are still in situ. This is a modification of a method first described to embed mineralised bone and involves the use of plastic embedding and a diamond saw. Using this technique, a permanent record is made of the tube location and patency.

A retrospective study of outcome of Mohs' micrographic surgery for cutaneous squamous cell carcinoma using formalin fixed sections.

The surgical management of recurrent or large squamous cell carcinoma (SCC) can be challenging as tumours often extend beyond visible margins. Micrographic surgery is a potentially effective method of ensuring complete clearance of tumour. A retrospective study of all cases of SCC treated by micrographic surgery in this department between 1986 and 1996 has been done. Sixty-one patients were treated using a formalin-fixed paraffin-embedded tissue technique with a median follow-up of 4 years. In two cases there was local recurrence and in three others metastasis to local lymph nodes. The overall cure rate was 92% (56 of 61), which compares favourably with published series using chemosurgery and frozen tissue techniques. The results show that this technique of micrographic surgery is a satisfactory and cost-effective alternative to conventional frozen section techniques in the treatment of SCC. The formalin-fixed tissue method has the advantage of providing high-quality permanent histological sections using existing conventional pathology services.

Receptor mechanisms mediating differentiation and proliferation effects of retinoids on neuroblastoma cells.

The aim of this study was to clarify retinoid receptor mechanisms mediating the effects of 9-cis retinoic acid (RA) and investigate the ability of RAR- and RXR-specific analogues to induce differentiation and inhibit proliferation in neuroblastoma cells. Differentiation and the inhibition of proliferation by 9-cis RA, but not all-trans RA, were inhibited by the RXR-homodimer antagonist LG745. The RXR-specific agonist LGD1069 was ineffective at inducing differentiation or inhibiting proliferation, but showed marked synergism with RAR-specific agonists with respect to inhibiting proliferation. These data suggest that the effects of 9-cis RA are mediated via both RXR-homodimers and heterodimers. However, combinations of RAR- and RXR-selective analogues were not as effective at promoting differentiation. This study indicates that different receptor mechanisms are involved in retinoid-induced differentiation and inhibition of proliferation in neuroblastoma cells.

Neoadjuvant chemotherapy with doxorubicin and cisplatin in malignant fibrous histiocytoma of bone: A European Osteosarcoma Intergroup study.

PURPOSE: Studies involving small case series have suggested that malignant fibrous histiocytoma of bone (MFH-B) is a chemosensitive tumor and that chemotherapy may improve survival. In this study, we evaluated clinical and pathologic response rates and survival in a series of patients treated with a consistent chemotherapy regimen of doxorubicin and cisplatin (DOX/DDP). PATIENTS AND METHODS: Study patients were required to have biopsy-proven MFH-B, no previous chemotherapy, and primary or metastatic measurable disease and to be /= 90% necrosis). Median time to progression was 56 months, and the 5-year progression-free survival rate was 56% (95% confidence interval [CI], 40% to 72%). Median survival time was 63 months, and the 5-year survival rate was 59% (95% CI, 41% to 77%). Patients with a good pathologic response had longer survival times and times to progression than did those with a poor response. Also treated were two patients with locally recurrent and nine with metastatic disease, and these patients had a median survival time of 17.5 months. CONCLUSION: Our study suggests that adjuvant or neoadjuvant chemotherapy with DOX/DDP is beneficial in MFH-B. Good pathologic response rates and survivals are quite comparable with those for osteosarcoma, a related bone tumor for which adjuvant or neoadjuvant chemotherapy is an accepted practice.

Differential effects of retinoic acid isomers on the expression of nuclear receptor co-regulators in neuroblastoma.

Retinoic acid modulates growth and induces differentiation and apoptosis of neuroblastoma cells in vitro, with the all-trans and 9-cis isomers having different biological properties. Transcriptional activation in response to retinoic acid isomers is mediated by retinoic acid receptors and retinoid X receptors. The differential expression of co-activators and co-repressors which preferentially interact with retinoic acid receptors or retinoid X receptors may be a mechanism leading to different cellular responses to 9-cis and all-trans retinoic acid. To test this hypothesis, we have studied the expression of the nuclear receptor co-regulators TIF1alpha, TIF1beta, SUG1 and SMRT in the N-type and S-type neuroblastoma cell lines SH SY 5Y and SH S EP. Transcripts for all four co-regulators were expressed in these neuroblastoma cells. The expression of TIF1alpha, TIF1beta and SUG1 did not change in response to retinoic acid; however, SMRT was induced in both neuroblastoma cell lines, but particularly by all-trans retinoic acid in SH S EP cells. An additional co-activator, Trip3, was isolated by differential mRNA display and shown to be preferentially induced by 9-cis retinoic acid in SH SY 5Y and SH S EP cells. These data suggest that retinoic acid isomer-specific induction of nuclear receptor co-regulators may determine, in part, the differential biological effects of retinoic acid isomers.

Bone morphogenetic protein 6 in skeletal metastases from prostate cancer and other common human malignancies.

Prostatic adenocarcinoma commonly metastasizes to bone. Unlike most other bony secondaries, the majority of skeletal prostatic metastases are osteoblastic rather than osteolytic in nature. Several growth factors which are known to stimulate bone formation are expressed in benign and malignant prostate cells, but none has been specifically linked to osteosclerotic metastases. Bone morphogenetic proteins (BMPs) induce ectopic bone formation in vivo. We have reported previously that BMP-6 mRNA and protein are expressed in the majority of primary prostatic carcinomas with established skeletal metastases but rarely in clinically organ-confined tumours. This study examines the expression of BMP-6 mRNA in matched prostatic primary and secondary bony lesions and in isolated skeletal metastases from prostatic adenocarcinomas, as well as other common human malignancies, by in situ hybridization. BMP-6 mRNA was detected in 11 out of 13 bone metastases from prostate carcinoma and in three paired samples of primary prostate carcinoma and matching skeletal metastasis. Weak signals for BMP-6 were also present in 5 out of 17 skeletal deposits from non-prostatic malignancies. BMP-6 mRNA appears to be strongly expressed in prostatic adenocarcinomas, both in the primary tumour and in bone metastases. It is also expressed, though less frequently, in skeletal metastases from other human carcinomas. Our findings suggest that BMP-6 may hold potential as an attractive marker and possible mediator of skeletal metastases, particularly in prostate carcinoma.

Mohs' surgery of periocular basal cell carcinoma using formalin-fixed sections and delayed closure.

Mohs' surgery of periocular basal cell carcinoma (BCC) ensures a high cure rate with maximal preservation of normal tissue. The formalin-fixed paraffin-embedded tissue technique allows Mohs' surgery to be performed using routine pathology facilities and permits the efficient use of operating room personnel and theatre time. The inevitable delay between excision and closure may potentially result in a poor functional and cosmetic outcome, particularly around the eye. We prospectively studied all patients with periocular BCC treated with this technique at our unit between 1985 and 1996. One hundred and twenty-three periocular BCCs in 120 patients were treated. Microscopic clearance was achieved in all cases. Closure was performed on average 5 days after the initial excisional stage. Closing procedures included direct closure, flaps and grafts. Significant complications affecting outcome were noted in only two patients. Eighty-eight per cent of patients assessed had a functional and cosmetic result regarded as excellent, good or adequate. Mohs' surgery of periocular BCC using formalin-fixed paraffin-embedded tissue and delayed closure results in a satisfactory functional and cosmetic outcome and offers a viable alternative to the frozen section fresh tissue technique.

Mononuclear phagocyte populations in the transplanted human lung.

BACKGROUND: Dendritic cells (DC) are essential for the development of alloreactivity, however, little has been published regarding the distribution and phenotype of these and related mononuclear cells in human lung transplantation. METHODS: Lung frozen sections were examined for the presence of CD1a+ DC and for mononuclear cells and alveolar macrophages expressing CD11b and CD68. The effects of transplantation and immunosuppression were assessed by comparison of normal transplant transbronchial biopsy specimens to specimens from unused donor lungs; the normal transbronchial biopsy specimens also were compared with those showing rejection or obliterative bronchiolitis. RESULTS: All biopsy specimens, including those with obliterative bronchiolitis, showed a marked depletion of CD1a+ DC in lung allografts. This has not been described previously. In addition, transplantation and immunosuppression reduced alveolar macrophage coexpression of CD68 and CD11b, and this was reversed in acute rejection. CONCLUSION: The roles of pulmonary DC and other mononuclear phagocyte subpopulations need to be further defined, and data from animal models of lung transplantation should be interpreted with caution.